In freezing of biological material, two freezing stages are recognized: nucleation and crystallization. In the first stage ice nucleation occurs in the solution outside the cells. In order to minimize cellular damage, it is critical to control during this stage (nucleation) both the interface velocity of the cold front and the direction of thermal gradient within the object. Normally, in some biological materials (e.g. blood, cell suspensions, plasma, semen and other liquid samples) the best survival is obtained when the freezing rate at this stage is relatively rapid (10° C./min or more). In other cases (e.g. organs or organ fragments), it is accepted that a slow freezing rate at this stage (0.5° C./min or less) would improve freezing.
The next stage is that of crystallization, an exothermic process that produces latent heat within the frozen material, causing a period of time when the biological material remains isothermal, or even experiences an increase in temperature: latent heat exudes from the biological material and thus, although the material is being cooled no temperature change is observed or the temperature may even rise. This in turn causes spontaneous freezing and thawing cycles which are hazardous to the biological material.
Permitting osmosis of water out of the cells at this stage would reduce damage to the cells, and the increase of intracellular concentration would cause the cells to vitrify rather than freeze. This is affected by the rate of freezing, and thus, in order to optimize the biological material's survival of this stage control of the rate of freezing is important. The optimal rate depends on the type and composition of the biological material being frozen.
In addition to the above, cryopreservation of material having a large volume (e.g. tissues, organs or portions thereof) is associated with heat transfer and mass problems that are not associated to the same extent with cryopreservation of isolated cells. For example, in conventional freezing methods, ice grows at an uncontrolled velocity and morphology and may disrupt and kill cells by mechanical destruction of the tissue architecture. Due to the large size of macroscopic material, large uncontrolled thermal gradients may develop from the surface of the sample to its interior.
One method that was devised to allow freezing biological material of a large volume is disclosed in U.S. Pat. No. 5,863,715. In this patent, the biological material is placed in a flexible container, such as a bag. The bag is then flattened in a holder that maintains an essentially constant cross-sectional area of the bag in order to minimize thermogradients. The holder is then cooled along with the bag contained therein.
It is well established that directional freezing, a process in which a cold front propagates in a controlled manner through the frozen object, improves the chances of biological material to survive freezing and thawing. In this process a temperature pattern (or gradient) is established in the object being frozen to form a propagation cold front within the object, resulting in improved chances of survival.
A successful method of directional freezing is disclosed in U.S. Pat. No. 5,873,254. In this patent, a freezing apparatus is used to establish a laterally varying thermal gradient and the biological sample is moved along the thermal gradient at a controlled velocity. Additional methods were developed in order to improve the freezing of large volume objects. For example, WO 03/056919 discloses freezing biological samples via an isothermal stage, wherein the temperature is changed until temperature of the sample in an outer zone equals intermediate temperature and changing temperature until the majority of the sample is in a final temperature. This method may be used in conjunction with directional freezing but is not limited thereto. Another process is disclosed in WO 03/020874 in which the biological sample is agitated during its migration along a thermal gradient.
A method for cryopreservation of a biopharmaceutical is disclosed in U.S. Pat. No. 6,337,205. The sample to be frozen is inserted into special oblong vials that have special appendages, termed “ice crystal-nucleating structures”, situated at the opposite ends of the vial's oblong cross-section. The vials are placed within a compartment of a cryopreservation apparatus, said compartment containing a cryopreservation fluid. A freezing front is then generated at one of the walls of the apparatus that is adjacent to one of the appendages, and propagates through the cryopreservation fluid. Due to the special shape of the appendage, nucleation begins at the appendage, and thus the cold front propagates within the sample in a direction that is away from the cooling wall and along the oblong cross section of the vial. In an alternative disclosed in U.S. Pat. No. 6,337,205, two cold fronts may be generated in the compartment, in opposing directions, by opposing walls of the apparatus. In this method, the freezing of the sample is achieved indirectly, in the sense that the cooling wall of the apparatus cools the cryopreservation fluid, which in turn cools the vial (and the sample within it).